Obesity has increased in prevalence worldwide, which is thought to be due to the influences of an obesity-promoting environment and genetic factors. Despite these influences there are individuals and animals that resist obesity. Spontaneous physical activity (SPA), which generates nonexercise activity thermogenesis (NEAT), can reduce weight gain. Injections of hypothalamic neuropeptide orexin A (OxA) into the lateral rostral hypothalamus (rLH) increased levels of SPA and increase obesity resistance. The neurological mechanism that drive SPA are not fully defined. Understanding the mechanism(s) by which NEAT is regulated will have the potential to develop novel approaches to preventing and treating obesity. OxA has been shown to activate extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen activated phosphate kinase (MAPK) in various cell models. Mice globally lacking the inhibitor for both these kinases, MKP-1, have been reported to be obesity resistant due in part to an increase in energy expenditure and SPA, but it is unknown whether OxA signaling is altered. OxA has also been recently shown to increase ATP and the transcription factor hypoxia inducible factor-1a (HIF-1a) in hypothalamic tissue under normoxic conditions. This is noteworthy given that HIF-1a increases oxidative phosphorylation. Additional independent studies have shown that HIF-1a expression is regulated in part by MAPKs and the transcriptional coactivator PGC-1a. PGC-1a is a regulator of mitochondrial biogenesis, can simultaneously upregulate genes that protect against oxidative stress, and increases ATP production. The central hypothesis proposed in this application is: SPA effects on weight gain depend in part on OxA signaling in the rLH, which alters genes and proteins involved in short- and long-term intracellular metabolic function. The three specific aims that will test this hypothesis are: 1) Determine in vitro if OxA increases HIF-1? by the activation of ERK1/2 and p38 MAPKs. 2) Test in vitro whether OxA-induced activation of HIF-1? mediates intracellular metabolic changes in rat rLH sections. 3) Evaluate in vivo if OxA treatment ameliorates or prevents changes in the ERK1/2-p38-MAPK-HIF-1?- PGC-1? pathway and intracellular metabolic responsiveness in the rLH following the development of diet-induced obesity. The immediate goal of the work proposed will characterize cellular mechanisms important in OxA-induced SPA using in vitro and in vivo models, with a long-term goal of developing therapeutic treatments for obesity through pharmacological manipulation of these identified pathways. The expected outcome is that OxA will increase the short- and long-term intracellular metabolic capacity of activated neurons, resulting in the maintenance of elevated SPA and obesity resistance. This will impact the field of obesity research by identifying a mechanism through which intracellular OxA signaling might contribute to obesity resistance. The innovation and the challenge to current paradigms are that the proposed studies suggest a new mechanism through which OxA elevates SPA, which is by altering genes that increase intracellular metabolic resistance in activated rLH neurons. This mechanism may be responsible for conferring long-term obesity resistance and would provide a new therapeutic target for obesity.